An unbiased screen identified the Hsp70-BAG3 complex as a regulator of myosin binding protein C3 stability.

Thompson, A. D.; Wagner, M. J.; Rodriguez, J.; Malhotra, A.; Vander Roest, S.; Lilienthal, U.; Shao, H.; Yob, J.; Prosser, B. L.; Helms, A. S.; Gestwicki, J. E.; Ginsburg, D. E.; Day, S. M.

doi:10.1101/2022.10.08.511444

bioRxiv · 10.1101/2022.10.08.511444

An unbiased screen identified the Hsp70-BAG3 complex as a regulator of myosin binding protein C3 stability.

Thompson, A. D.·Wagner, M. J.·Rodriguez, J.·Malhotra, A.·Vander Roest, S.·Lilienthal, U.·Shao, H.·Yob, J.·Prosser, B. L.·Helms, A. S.·Gestwicki, J. E.·Ginsburg, D. E.·Day, S. M.

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Abstract

Structured AbstractO_ST_ABSObjectiveC_ST_ABSWe aim to identify regulators of myosin binding protein C3 (MyBP-C) protein homeostasis. BackgroundVariants in myosin binding protein C3 (MYBPC3) account for approximately 50% of familial hypertrophic cardiomyopathy (HCM). Most pathogenic variants in MYBPC3 are truncating variants that lead to reduced total levels of MyBP-C protein. Elucidation of the pathways that regulate MyBP-C protein homeostasis could uncover new therapeutic strategies that restore normal protein levels. MethodWe developed a high-throughput screen to identify compounds that can increase or decrease steady-state levels of MyBP-C in an induced pluripotent stem cell cardiomyocyte (iPSC-CM) model derived from a patient with HCM. To normalize results, we also monitored effects on myosin heavy chain (MYH) and focused on those molecules that selectively modulated MyBP-C levels. ResultsScreening a library of 2,426 known biologically active compounds, we identified compounds which either decreased (241/2426, 9.9%) or increased (29/2426, 1.2%) MyBP-C/MYH levels. After a rigorous validation process, including a counter screen for cellular toxicity, two compounds (JG98 and parthenolide) were confirmed as decreasing MyBP-C levels and no compounds were confirmed to increase MyBP-C levels. For further studies, we focused on JG98, which is an allosteric modulator of heat shock protein 70 (Hsp70), inhibiting its interaction with BAG domain co-chaperones. We found that genetic reduction of BAG3 phenocopies treatment with JG98 by reducing MyBP-C protein levels. ConclusionAn unbiased compound screen identified the Hsp70-BAG3 complex as a regulator of MyBP-C stability. Thus, approaches that stimulate this complexs function may be beneficial in the treatment of HCM. Highlights- Hypertrophic cardiomyopathy (HCM) is commonly caused by pathogenic MYBPC3 variants that reduce total wild-type MyBP-C (the protein encoded by MYBPC3). - It is critical to understand the regulators of MyBP-C protein homeostasis to uncover novel therapeutic strategies. - We developed and executed a high-throughput chemical screen in iPSC-CMs to identify compounds which alter steady-state levels of MyBP-C protein, revealing two compounds, JG98 and parthenolide, that significantly reduced MyBP-C levels. - Validation studies suggest that the complex between heat shock protein 70 (Hsp70) and its co-chaperone BAG3 is a dynamic regulator of MyBP-C stability, suggesting that this axis could be a new therapeutic target for HCM.

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Thompson, A. D., Wagner, M. J., Rodriguez, J., Malhotra, A., Vander Roest, S., Lilienthal, U., Shao, H., Yob, J., Prosser, B. L., Helms, A. S., Gestwicki, J. E., Ginsburg, D. E., Day, S. M.. 2022-10-08. An unbiased screen identified the Hsp70-BAG3 complex as a regulator of myosin binding protein C3 stability.. https://doi.org/10.1101/2022.10.08.511444

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